Normal human peritoneal fluids contain macrophage in different stages of maturation ranging from newly arrived peroxidase positive monocyte type cells to large acid phosphatase positive macrophages. This heterogeneity presents a problem when analyzing functional activity, as the expression of any particular function by a subpopulation of macrophages may be in balance and under the regulation of one or more other macrophage subpopulations. In this proposal different fractionation procedures will be applied to separate macrophages into subpopulations based on size and density. The subpopulations will be closely characterized for a variety of enzyme, antigen and receptor markers using a flow cytofluorometric approach allowing for simultaneous charactrization of cell size and three markers defined by red, blue and green fluorescence. A number of macrophage functions will be assayed on the more homogenous, defined subpopulations. In parallel, in vitro differeniating monocyte cultures will be similarly defined by flowcytofluorometry and the influence of biological stimuli on markers and functions assessed. The heterogeneity in peritoneal macrophage populations also provides an in vivo example of human monocyte to macrophage maturation. It is not known, however, whether monocytes follow a common pathway or whether different signals create distinct pathways or inhibit maturation. Peritoneal macrophage subpopulations will be followed under in vitro conditions to investigate if they go through the same changes as in vitro monocyte derived macrophages with similar marker profiles. The influence of several biological stimuli on monocyte to macrophage maturation will also be investigated. The results of these studies will show 1) if changes in any particular marker or combination of markers is associated with a certain function, 2) if differences in sensitivity to activation/stimulation by biological agents exist between maturing macrophages and 3) if signals in vivo direct the pathway marophages mature and whether it is irreversible, 4) if monocyte to macrophage development under various stimuli occurs, along a common pathway or a series of distinct differentiation pathways.